Settlement assays—laboratory studies

The antifouling activity of extracts of the seagrass Zostera marina were examined for effects on the settlement of a marine bacterium isolated from its surface (Todd et al. 1993). In this assay, extracts and pure compounds dissolved in methanol were coated onto the frosted ends of glass microscope slides. Controls consisted of slides coated with methanol. The solvent was then evaporated and the slides placed into 50-ml plastic tubes containing a suspension of Acinetobacter sp. (10 cells/ml). The tubes were incubated on a rotary shaker and every 20 min for 4 h slides were removed, stained with Hoechst (no. 2287, Sigma Co.) and the number of cells attached to surfaces at various extract concentrations counted by epifluorescence microscopy. This study was important in that it established a connection between antifouling activity and chemical compounds isolated from the seagrass. 

A similar assay was used to examine the effects of extracts from three soft corals Alcyonium paessleri, Clavularia frankliniana, and Gersemia antarctica, against three species of marine bacteria (Slattery et al. 1995). In this assay, replicate 20-g soft coral samples were extracted in 200 ml of solvent and then diluted to tissue level concentrations. Twenty-five microliters of each soft coral extract or a solvent control were then spotted onto glass microscope slide cover-slips. The coverslips were placed into individual wells of tissue culture plates that contained 2 ml of sterile-filtered seawater seeded with 50 Lil of a bacterial suspension (7.5 x 10 cells/ml). Following a 48-h incubation, the coverslips were washed with sterile-filtered seawater to remove unattached cells and stained with DAPI (4,6-diamidino-2-phenylindole). The number of cells in each of five replicate microscope fields per coverslip were counted using epifluorescence microscopy. Bacterial attachment was found in this study to be inhibited by the chloroform extracts of both A. paessleri and G. antarctica and the methanol extract of G. antarctica. The active chloroform extracts did not display antimicro- 

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